1. Field of the Invention
This invention relates to a DNA sequence which comprises a DNA fragment consisting of a region involved in the expression of a gene coding for an extracellular enzyme of a bacterium of the genus Bacillus and a region involved in the expression of the enzyme thus expressed and, if necessary, a DNA segment joined to said DNA sequence on its downstream side which includes a restriction endonuclease cleavage site or sites capable of joining thereto a DNA fragment containing a gene coding for a desired protein; an expression-secretion vector formed by joining the DNA sequence to a plasmid DNA or phage DNA capable of replicating in a host bacterium of the genus Bacillus; and a method of producting proteins by use of said vector.
2. Description of the Prior Art
The marked development of molecular biology in recent years has made it possible to produce and secrete a desired protein from a host microorganism by joining a gene coding for a desired protein to a proper vector and introducing the resulting recombinant DNA into a host microorganism.
As the host microorganism, Escherichia coli has been so far used which is most advanced in genetical and molecular-biological investigations. However, when Escherichia coli is used as the host bacterium, the desired protein is usually accumulated within the cells, thus making it very difficult to purify the desired protein. Furthermore, the desired protein, though purified, is unavoidably contaminated with pyrogens, raising a serious problem.
On the other hand, bacteria of the genus Bacillus have characteristics of extracellular secretion of a large amount of protein, lack pathogenicity and have long been used in the fermentation industry. Since their safety is thus established, they have been regarded as important as the host bacteria capable of solving the aforesaid problems involved in the separation and purification of proteins from the interior of the cells. Accordingly, it would be of great industrial significance from the viewpoint of microbial production of proteins to create an expression-secretion vector suitable for use in bacteria of the genus Bacillus.
From this point of view, attempts have been made to develop expression-secretion vectors capable of utilizing bacteria of the genus Bacillus as the host. Further, there is an increasing demand for expression-secretion vectors which permit an extracellular production of a desired protein in large amounts.